The proposed research builds on recent extensions to our hardware and software that allows, in a generalized resolution overlapping fashion, 3-dimensional reconstructions of subcellular structures at both the light and electron microscopic levels. We apply this methodology to the analysis of higher-order chromosome structure and function using the unique interphase and anaphase chromosomes of Drosophila melanogaster. New technology utilizing structured illumination optical microscopy (SIM) and electron microscopy tomography (EMT) allows novel new insights into 3-dimensional chromosome structure. A 3-dimensional Bar Code provides for a systematic approach to the study of 3 dimension diploid interphase chromosome topography in the nucleus on a cell-by-cell basis. Chromosome dynamics, in living nuclei, can now be studied using green fluorescent protein (GFP) to label specific chromosomal loci. The specific aims of this proposal are: A) Determine the 3 dimensional diploid chromosome topography in Drosophila early embryo nuclei using the 3-dimensional Bar Code methodology. B) Study the 4-dimensional dynamics of diploid chromosome behavior using the newly available GFP chromosome locus labeling methodology. C) Continue the determination of the 3-dimensional structure of anaphase chromosomes using SIM and EMT and their extensions.